Tropomyosin
Tropomyosins are a class of coiled-coil, rod like proteins, which as a homodimer, bind along actin filaments (1). Tropomyosin has been extensively studied in the context of muscle cells, where it regulates sarcomere contraction by inhibiting type II myosin from binding actin in the absence of Ca2+ (1-3). However, the role tropomyosins play in regulating actomyosin interactions in non-muscle cells is less defined, though several have been implicated in playing a role in the transformation process of metastatic cancers (4). Significance Though each class of myosin relies on the same actin substrate to generate force, a high level of specificity with regard to the type of myosin binding to each actin structure within the cell has been observed. (1) Recent studies have demonstrated that tropomyosins are able to provide a level of selectivity in the form of controlling which type of myosin motors are able to interact with each class of actin structures. (1, 4) Eukaryotic organisms depend upon these specific actomyosin interactions for diverse cellular functions, including: maintaining structural integrity, providing intracellular transport, and generating the contractile force necessary for endocytosis and division. The numerous possible combinations of more than twenty separate classes of myosin with over 40 isoforms of tropomyosin provide higher eukaryotic cells a mechanism in which to achieve the required actomyosin specificity. (1, 4) Applications The use of recombinant tropomyosin protein allows researchers the ability to evaluate the role of tropomyosin -- in the context of actin filament decoration -- through in vitro experimnenbts, such as bulk myosin ATPase assays, as well in vivo studies in which isoform specific roles of tropomyosin are gleaned from knockout and rescue experiments. Plasmid Construct A Tm5NM1 construct is used to over-express the acetylated-mimicking form of the protein in E. coli. The tropomyosin gene was PCR amplified from human cDNA using the following primers: 5’ NdeI-Tm5NM1 CAATCATATGGCTAGCATGGATAAGCTTAGAGAG (the NdeI site is italicized, the Tm5NM1 start codon is underlined) and 3’ BamHi-Tm5NM1 CAATGGATCCCTACAAGTCCTCAAGAGCTT (the BamHI site is italicized, and the Tm5NM1 stop codon is underlined). The 5’ primer includes an Ala-Ser (acetylation-mimicking) dipeptide immediately upstream of the Tm5NM1 start codon. The start codon for the recombinant protein actually lies in the second half of the NdeI site allowing for incorporation of the Ala-Ser codons. Each construct was individually cloned into the pET3a vector and confirmed by DNA sequencing. Protein Purification Tm5NM1 constructs were produced from E. coli BL21 DE3 competent cells. Cultures were grown in LB at 37 ͦC and induced by the addition of 0.4mM IPTG. Cells were harvested following and overnight expression of constructs conducted at 25 ͦC. Proteins were purification as previously described (5). Briefly, cell pellets were resuspended in Tm lysis buffer (100 mM NaCl, 10 mM imidazole, pH 7.2, 2 mM EDTA, 1 mM DTT) and lysed by sonication. The lysates were boiled for 10min while stirring and pelleted at 20,000 for 15 minutes to remove denatured proteins and other debris. Soluble tropomyosin in the supernatant was precipitated by bringing the pH down to 0.2 units below the isoelectric point for each construct, and again spun at 20,000 g for 15 minutes. The precipitated pellets were then resuspended in 50 mM NaCl, 10 mM imidazole, pH 7.4, 1 mM DTT, and 5 ug/mL leupeptin) and dialyzed for three rounds against the same buffer. References 1. Gunning PW, Schevzov G, Kee AJ, & Hardeman EC (2005) Tropomyosin isoforms: divining rods for actin cytoskeleton function. Trends in cell biology 15(6):333-341. 2. Cooper JA (2002) Actin dynamics: tropomyosin provides stability. Current biology : CB 12(15):R523-525. 3. Gordon AM, Homsher E, & Regnier M (2000) Regulation of contraction in striated muscle. Physiological reviews 80(2):853-924. 4. Wang CL & Coluccio LM (2010) New insights into the regulation of the actin cytoskeleton by tropomyosin. International review of cell and molecular biology 281:91-128. 5. Hodges AR, Bookwalter CS, Krementsova EB, & Trybus KM (2009) A nonprocessive class V myosin drives cargo processively when a kinesin- related protein is a passenger. Current biology : CB 19(24):2121-2125.